Eukaryotic translation initiation factors control several steps in the assembly of mRNA with 40S ribosomal[unreadable] subunits at the start of protein synthesis. Two of these factors, elF2 and elF3, are almost universally[unreadable] required for cap-dependent and cap-independent initiation. Eukaryotic initiation factor elF2 is a heterotrimer[unreadable] responsible for bringing initiator tRNA to ribosome-mRNA assemblies, and represents one of the key targets[unreadable] of negative translation regulation by kinases. Initiation factor elF3 includes at least twelve non-identical[unreadable] subunits and is required for translation of most m7G-cap- and IRES-driven mRNAs. The internal ribosome[unreadable] entry site (IRES) of hepatitis C virus (HCV) utilizes both elF2 and elF3 during translation initiation, and a[unreadable] direct contact between the IRES and elF3 is essential for efficient viral translation. This proposal aims to[unreadable] understand the biochemical and structural basis for the activities of these factors. We will develop methods[unreadable] for large-scale purification of these factors and will analyze their composition both alone and within functional[unreadable] ribosomal complexes defined by the HCV IRES using quantitative mass spectrometry. We will determine the[unreadable] role of elF3 in HCV IRES-mediated translation initiation by isolating and analyzing translation complexes that[unreadable] bind the wild type and mutant forms of the IRES in vitro and in vivo. Finally, we plan to determine molecular[unreadable] structures of elF2 and elF3 using both X-ray crystallography and electron microscopy, providing a structural[unreadable] framework for interpreting interactions that occur during both IRES-mediated and cap-dependent translation.[unreadable] Direct collaboration and interactions with the Gate, Harris, Hershey and Sarnow laboratories, as well as the[unreadable] proposed Mass Spectrometry Core Laboratory to be directed by Prof. Leary, is an integral part of these[unreadable] experiments.